With this proposal, we will test the overall hypothesis that regulation of cAMP levels in microdomains of signaling, rather than on the global scale, determines the function of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) in health and disease. The CFTR is a cAMP-stimulated anion channel that is critical for ion- and water homeostasis across many epithelia including in the airways and the gastrointestinal tract. CFTR hypofunction, as a result of mutations in the CFTR gene in Cystic Fibrosis (CF), or smoke exposure in Chronic Obstructive Pulmonary Disease (COPD), results in reduced levels of this channel in the apical membrane of epithelia which causes abnormal fluid and electrolyte transport. It is well established that CFTR function is stimulated by PKA phosphorylation. However, stimulating CFTR-dependent ion transport in CF model systems by increasing global cellular cAMP to supra-physiological levels with a combination of adenylyl cyclase activators and broad-spectrum PDE inhibitors has been met with mixed results and successes in the past. With this research proposal, we wish to test two novel approaches to overcome this limitation. Specific Aim 1 of this proposal will test the hypothesis that specific isoforms of cyclic nucleotide phosphodiesterases (PDEs), the enzymes that degrade and inactivate cAMP, are physically tethered to signaling complexes involving the CFTR and control the activity of this channel in compartmentalized microdomains of cAMP signaling. We wish to test the idea that inactivation of this localized pool of PDE activity or its displacement from CFTR signaling complexes provides a safer and more effective approach to stimulate CFTR function compared to increasing global cAMP levels, as the latter approach induces significant cellular feedback responses and undesirable side effects that ultimately limit its efficacy. The human genome encodes for 21 PDE genes, which are likely expressed as more than 100 protein variants. Inhibitors of type 4 PDEs (PDE4s) have been shown to stimulate CFTR in immortalized epithelial cell lines. We have now generated preliminary data showing that PDE4 isoforms are the major regulators of CFTR activity in primary lung epithelial cells and are physically tethered to this channel suggesting that these are the PDEs that control cAMP in CFTR microdomains. In Specific Aim 1 of this proposal, we will characterize the interaction of CFTR with individual PDE4 isoforms and test the effect of selective inactivation of these PDE4s on CFTR function in non-CF and CF cells and tissues. In Specific Aim 2 of this proposal, we will generate novel FRET-based biosensors that can measure cAMP levels and PKA activity in the immediate vicinity of the CFTR. This will allow us to trace the pools of cAMP that determine CFTR function, identify the key regulators in this compartment, and explore possible differences in cAMP signaling events between CF and non-CF epithelia.